MOLECULAR AND CELLULAR BIOLOGY, Feb. 1991, p. 979-986
`0270-7306/91/020979--08$02.00/0
`Copyright © 1991, American Society for Microbiology
`
`Vol. 11, No. 2
`
`Regulation of Phosphorylation of the c-erbB-2/HER2 Gene Product
`by a Monoclonal Antibody and Serum Growth Factor(s)
`in Human Mammary Carcinoma Cells
`RAK.ESH KUMAR,1* H. MICHAEL SHEPARD,2 AND JOHN MENDELSOHN1·3
`Laboratory of Receptor Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York,
`New York 100211; Developmental Biology, Genentech Inc., South San Francisco, California 9408a2;
`and Cornell University Medical College, New York, New York 100213
`
`Received 9 May 1990/Accepted 17 November 1990
`
`Monoclonal antibody (MAb) 4DS was used to analyze the phosphorylation of p185HER2, the gene product of
`c-erbB-21HER2, in SK-BR-3 cells. Culture in the continuous presence of 4DS reduced the in vivo steady-state
`levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4DS may have
`interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of
`p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were
`grown under serum-free conditions, the steady-state levels ofpl85HER2 phosphorylation were reduced by 56%,
`and addition of 4DS further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure
`to increasing concentrations of newborn calf serum in these cultures, the.re was a linear increase in
`tyrosine-specific phosph~lation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum.
`Phosphorylation of pl85H R2 in the presence of newborn calf serum was not attributable to stimulation of the
`epidenna1 growth factor receptor by epiderma1 growth factor or by transforming growth factor-a. Extension
`of these observations to two other mammary carcinoma ceU lines, MDA·MB-453 and BT-474, a1so demon(cid:173)
`strated a significant capacity of serum to induce pl85HER2 phosphorylation. The demonstration of antibody·
`mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma
`cells may also produce and secrete a factor or factors which may activate l185HER2. Our observation that
`growth-inhibitory MAb 4DS is able to reduce the phosphorylation of p185HB by newborn calf serum and by
`a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2
`as a signal transduction pathway to regulate ceU proliferation.
`
`Proto-oncogenes are a group of normal genes which play
`important roles in the regulation of cell proliferation and
`function (2, 5). Abnormalities in the expression , structure, or
`activity of proto-oncogene products contribute to the devel(cid:173)
`opment and maintenance of the malignant phenotype in
`complex but important ways (36, 37, 46). Evidence that the
`gene products of several activated proto-oncogenes are
`either growth factors or growth factor receptors has sug(cid:173)
`gested a possible link between proto-oncogenes and growth
`factors (20). For example, the receptor for macrophage
`colony-stimulating factor is identical to the product of c-fms
`(35), and c-erbB-1 encodes the receptor for epidermal growth
`factor (13) and transforming growth factor-a (TGF-a) (43).
`Growth factor receptors encoded by proto-oncogenes are
`transmembrane glycoproteins with intrinsic tyrosine-kinase
`activity (22). Receptor tyrosine kinases are activated by
`binding of their respective ligands, the growth factors (48).
`This activity is thought to be an integral part of signal
`transduction processes involved in the regulation of cell
`proliferation (21). Overexpression of some growth factor
`receptors has been shown to induce transformed properties
`in recipient ceUs (11, 32), possibly because of excessive
`activation of signal transduction mechanisms. Furthermore,
`a number of tumor cells with increased expression of growth
`factor receptors also produce ligands for these receptors
`(10).
`HER2 (also known as c-erbB-2 or c-neu), the human
`
`• Corresponding author.
`
`979
`
`homolog of the rat proto-oncogene neu (9), encodes a
`185-kDa transmembrane glycoprotein with intrinsic tyrosine
`kinase activity which is presumed to be the receptor for an
`as-yet-unidentified ligand (3, 39). pl85HERz also has homol(cid:173)
`ogy to, but is distinct from , the epidermal growth factor
`receptor (EGF-R), which is the product of c-erbB-1. Both
`proteins have a cysteine-rich extracellular domain, a trans(cid:173)
`membrane domain, and an intracellular tyrosine kinase (4,
`31 , 47). In spite of sequence homology between c-erbB-2 and
`c-erbB-1, EGF does not bind to pl85HERz (33). pl85HERz has
`been shown to be overexpressed or amplified or both in a
`number of human malignancies: breast (45), ovarian (38),
`thyroid (1), lung (7), salivary gland (34), and stomach (50). In
`addition, pl85HER2 is a potent oncogene capable of inducing
`transformation and tumorigenesis when overexpressed in
`NIH 3T3 ceUs (12, 19). Overexpression of pl85HER2 also
`induces tumor cell resistance to macrophage killing (15).
`Thus p185HERZ may have an important role in the develop(cid:173)
`ment and maintenance of human tumors.
`These observations suggest that receptor-associated ty(cid:173)
`rosine kinase activity of overexpressed proto-oncogene pro(cid:173)
`tein products is important for the regulation of cell growth.
`We have developed a panel of monoclonal antibodies
`(MAbs) reactive with domains of the human EGF-R (23) and
`p185HER2 (17, 18) in intact ceUs and have demonstrated
`antiproliferative effects of these antibodies in vitro (16, 18,
`23) and in vivo (29). Antibody 405 , which is specifically
`directed against pl85HERZ, exhibits strong antiproliferative
`activity on cultured human breast tumor cell lines which
`overexpress pl85HER2 (18). Since p185H.ER2 is a receptor
`
`1 of 8
`
`Celltrion, Inc., Exhibit 1088
`
`

`

`980
`
`KUMAR ET AL.
`
`MOL. CELL. BIOL.
`
`with intrinsic tyrosine kinase activity, we investigated the
`modula'.i-Jn of p185HERi phosphorylation by MAb 405. We
`report here that activation of phosphorylation of p185HERi
`by serum was reduced in the presence of an excess of MAb
`405 and that MAb-sensitive phosphorylation was mediated
`by a growth factor or factors other than TGF-o: or EGF.
`Furthermore, SK-BR-3 cell-conditioned medium contained a
`factor(s) that could activate pl85HERi phosphorylation and
`was partially inhibited by MAb 405 .
`
`MATERIALS AND METHODS
`
`Materials. MAbs 4D5 (18) and 906 (44) were raised against
`human p185HER2
`• MAbs 528 and 225 bind to the human
`EGF-R \23). Antiphosphotyrosine MAb PY-69 was obtained
`from ICN Biochemicals, Inc. Rabbit immunoglobulin to
`mouse immunoglobulins G (RAM) was supplied by Accurate
`Chemicals, Westbury, N.Y. 32P; (carrier free ; 28.5 Ci/nmol)
`and 35S-labeled L-cysteine (1,030 Ci/mmol) were purchased
`from New England Nuclear, Boston, Mass.
`Cell lines and cell culture. Human breast tumor cell lines
`SK-BR-3, BT-474, and MDA-MB-453 were obtained from
`the American Type Culture Collection. The A431 human
`epidermal carcinoma cell line was originally supplied by
`Gordon Sato. All cell lines except MDA-MB-453 (which was
`grown in L-15 medium) were maintained in Ham F-12-
`Dulbecco modified Eagle medium (1:1, vol/vol) (F-12/
`DMEM) supplemented with 10% fetal bovine serum.
`Labeling of p185HER2 with 32P 1 and [35S)cysteine. Cells (3 x
`105
`) were plated in F-12/DMEM in each well of a six-well
`dish. Twenty-four hours later, cultures were washed with
`phosphate-free medium and incubated for up to 15 h in
`phosphate-free F-12/DMEM containing 0.4 mCi of 32P; per
`ml in the presence or absence of MAb and newborn calf
`serum. At desi.red times, cells were harvested in 400 µI of
`lysis buffer (20 mM HEPES [N-2-hydroxyethylpiperazine(cid:173)
`N' -2-ethanesulfonic acid; pH 7.5), 1% Triton X-100, 10%
`glycerol, 1.5 mM magnesium chloride, 1 mM ethyleneglycol
`bis-N,N,N' ,N'-tetraacetic acid, 0.1 mM phenylmethylsulfo(cid:173)
`nyl fluoride , 10 µg of leupeptine per ml , 2 mM sodium
`orthovanadate) at 4°C for 20 min. The lysate was centrifuged
`at 10,000 rpm in an Eppendorf microfuge for 10 min, and
`then 60 µl of Pansorbin was added as described elsewhere
`(42). For labeling with [35S]cysteine, the cells were washed
`with cysteine-free medium and refed with cysteine-free
`F-12/DMEM containing 0.15 mCi of [35S]cysteine per ml
`with or without 5% newborn calf serum.
`lmmunoprecipitation and SDS-polyacrylamide gel electro(cid:173)
`phoresis. Aliquots (350 µI) of the cell lysates (or equal
`amounts of trichloroacetic acid-precipitable counts per
`minute) containing 32P-labeled or [35S]cysteine-labeled
`p185HER2 were subjected to immunoprecipitation with 10 µg
`of MAb 906, 528, or PY-69 at 4°C for 2 h. Immune
`complexes were collected by absorption to RAM-protein
`A-Sepharose beads at 4°C for 1 h. Beads were washed three
`times with 1 ml of buffer (20 mM HEPES [pH 7 .5], 150 mM
`NaCl, 0.1% Triton X-100, 10% glycerol, 2 mM sodium
`orthovanadate). Washed pellets were mixed with 40 µI of
`sample loading buffer (10 mM Tris HCl [pH 6.8], 1% sodium
`dodecyl sulfate [SOS], 0.2% 2-~-mercaptoethanol, 10% glyc(cid:173)
`erol, 0.001% bromophenol blue), heated at 95°C for 5 min,
`and resolved on a 7% SDS-polyacrylamide slab gel (26). The
`efficiency of precipitating labeled receptor with MAb 906 is
`80 to 90% when this procedure is used. Low-molecular-mass
`colored markers (Amersham Corp.) were used as standards.
`Phosphoamino acid analysis. The band corresponding to
`
`2 3 4 5
`
`FIG. 1. Effect of MAb 405 on steady-state levels of pl85HERZ
`phosphorylation in SK-BR-3 cells. Subconfluent cultures were la(cid:173)
`beled with 32P; (400 µCi in 1 ml of phosphate free F-12/DMEM
`supplemented with 5% newborn calf serum) in the continuous
`presence of different amounts of antibody for 15 h. Detergent
`extracts were made, and pl85HER2 was immunoprecipitated by using
`MAb 9G6 and then resolved by 7% SDS-polyacrylamide gel elec(cid:173)
`trophoresis (Materials and Methods). An autoradiogram resulting
`from 16 h of exposure of the dried gel is shown here. The arrow
`indicates the position of 32P-labeled pl85HER2 • Lane 1, Control cells;
`lanes 2 to 4, cells treated with MAb 405 at 30, 150, and 300 nM,
`respectively; lane 5, cells treated with 400 nM F(ab) fragment of
`MAb 405. The amounts (in counts per minute) of pl85HER2 in each
`lane were 4,453 (lane 1), 1,967 (lane 2), 1,785 (lane 3), 1,040 (lane 4).
`and 335 (lane 5). Counts were corrected by subtracting the back(cid:173)
`ground of 60 cpm. The results shown are representative of results in
`six different experiments.
`
`the 185-kDa HER2 protein, resolved as described above,
`was excised out of the gel. 32P-labeled pl85HERz in a gel slice
`was partially hydrolyzed with 200 µI of 6 N HCI at ll0°C for
`1 h. Two portions (10 µleach) of the hydrolysate were taken
`for measurement of radioactivity in a liquid scintillation
`counter to determine the total incorporation of 32P into the
`pl85HER2 receptor. The rest of the hydrolysate was dried,
`suspended in distilled water, and applied to a Dowex
`AG1-X8 column. The column was washed with distil.led
`water, and the absorbed 32P-labeled materials were eluted
`with 0.5 N HCI and lyophilized. The recovery of radioactiv(cid:173)
`ity by this procedure was 78 to 85%. 32P-phosphoamino
`acids mixed with unlabeled carrier phosphoamino acids
`(phosphoserine, phosphoth.reonine, and phosphotyrosine
`[1:1:1)) were analyzed by thin-layer electrophoresis as de(cid:173)
`scribed elsewhere (8).
`
`RESULTS
`MAb 405 reduces amount of 32P-labeled pl8SH£R2
`• MAb
`4D5 was used to investigate the regulation of phosphoryla(cid:173)
`tion. SK-BR-3 cells, which have an amplified c-erbB-2 gene
`(45), were cultured for 15 h in medium containing 32P; in the
`continuous presence of various concentrations of MAb 4D5.
`The pl85/fERi from these cells was immunoprecipitated with
`another anti-p185H£Ri MAb, 906, which recognizes a dis(cid:173)
`tinct epitope of p185HERz, and resolved by SOS-polyacryl(cid:173)
`amide gel electrophoresis. Results of such an experiment are
`shown in Fig. 1. Treatment of cells with 4D5 reduced in vivo
`steady-state levels of 32P-labeled p185HERi up to 80% in a
`dose-dependent manner (lanes 2 through 4). There was 49%
`± 8% reduction in phosphorylation by 150 nM MAb 405 in
`eight different experiments. When the F(ab) fragment of 4D5
`was used instead of intact antibody, comparable or greater
`reduction of 32P-labeled p185HER2 was observed (lane 5). As
`a control, SK-BR-3 cells were incubated with another MAb,
`225 lgGI, specifically directed against the EGF-R, and there
`was no effect on the amount of 3 2P-labeled pl85He:Ri (un(cid:173)
`published data). The reduction in steady-state levels of
`32P-labeled p185H£RZ was not due to interference by 405
`with MAb 906 during the immunoprecipitation reaction, as
`immunoprecipitation performed with another polyclonal an-
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 2 of 8
`
`2 of 8
`
`Celltrion, Inc., Exhibit 1088
`
`

`

`VOL. 11, 1991
`
`MODULATION OF PHOSPHORYLATION OF pl85mlU
`
`981
`
`Abs
`
`P185
`2 3 1
`
`____ ...
`
`EGF-R
`l' 21 31
`
`I
`
`--
`
`FIG. 2. Specificity of the reduction of 32P-labeled pl85H£Rl by
`MAb in SK-BR-3 cells in the presence or absence of MAb 405.
`Subconfluent cells were labeled with 32Pi for 15 h. The cells were
`lysed in 600 µJ of extraction buffer and divided into two equal pans
`of 250 µ.I each. lmmunoprecipitation was performed with anti-pl85
`MAb (lanes l to 3) or with anti-EGF-R MAb 528 (lanes l ' to 3'). An
`autoradiogram of a dried gel is shown here. Lane l and l '. Control:
`lanes 2 and 2'. 30 nM MAb 405: lanes 3 and 3'. 150 nM MAb 405.
`Counts per minute: lane 1. 5.985; lane 2, 3,798; lane 3, 3,120; lane l '.
`853; lane 2', 779; lane 3'. 932. Abs, Antibodies.
`
`tibody (18) recognizing the carboxy-terminal 17 amino acids
`of pl85HER2 gave similar results (unpublished data).
`Next , we examined the possibility of general inhibitory
`effects of MAb 405 on the steady-state levels of other
`32P-labeled receptor proteins by analyzing the amount of
`32P-labeled pl85HER.Z and 32P-labeled EGF-R in the same
`experiment (Fig. 2). These results indicated that there was
`no reduction of 32P-labeled EGF-R during 15 h of treatment
`of SK-BR-J cells with 150 nM MAb 405. which had reduced
`the amount of 32P-labeled pl85HER by 48%.
`Analysis of reduction of pl8SH£Rz phosphorylation. The
`reduction of steady-state levels of 32P-labeled pl85HERi by
`MAb 405, shown in Fig. 1 and 2, could result from down(cid:173)
`regulation of pl85HER2 and/or interference in the activation
`of pl85HER2 phosphorylation by a direct or indirect m1:cha(cid:173)
`nism(s). In initial studies to explore these possibilities.
`parallel cultures of cells were metabolically labeled with
`[35S)cysteine or 32P;. During 11 h of concurrent incubation
`with MAb 405 , there was a 45% reduction in 32P-labeled
`pl85HERZ (Fig. JA) and only a 14% reduction in JSS-labeled
`pl85HERZ (Fig. JB). This suggests that the reduced 32P label
`in pl85HERZ in the presence of MAb 405 can only partially
`be attributed to reduced pl8511ERZ content. Next, we per(cid:173)
`formed a similar experiment comparing the capacities of the
`monovalent F(ab) fragment of MAb 405 and an intact MAb
`405 to affect the reduction of 3~S-labeled pl85HERi . There
`was no change in 35S-labeled pl85HERi in the presence of
`F(ab), but there was a 26% reduction caused by MAb 405
`(Fig. JC, lanes J and 2, respectively). The results obtained in
`the immunoprecipitation experiments documented in Fig.
`JA through C were confirmed by immunoblotting (0 ). lmmu(cid:173)
`noblotting of the 32P-labeled SK-BR-J cell extracts used in
`Fig. 2 demonstrated only a marginal reduction in the content
`of pl85HERz protein when cells were cultured in the presence
`of MAb 405 but a substantial reduction in the amount of
`32P-labeled pl85HERZ (Fig. 2). The expression ofEGF-R was
`not affected. Immunoblotting of similar unlabeled SK-BR-J
`extracts also demonstrated very little reduction in the con(cid:173)
`tent of pl85HERi by MAb 405 (Fig. J D, experiment 2).
`These findings indicate that increased receptor catabolism
`induced by a MAb cannot fully account for the observed
`reduction in 32P labeling and show £with F(ab)) that reduced
`labeling is dissociated from reduced content of pl85HER~ .
`Next we addressed the possibility that the reduction in
`32P-labeled pl85HER.Z associated with exposure to MAb 405
`could be related to a change in the level of expression of
`pl85HERi on the plasma membrane or to the extent of
`
`A.
`
`8.
`
`C.
`
`405
`0.
`
`+
`
`EXP 1
`
`Abs P185
`
`EGF-R
`
`+
`
`2
`
`3
`
`EXP. 2
`Pl85
`
`-
`
`- +
`+
`+
`405
`FIG. 3. Analysis of the reduction ofpl85H£Ri phosphorylation in
`SK-BR-3 cells treated with MAb 405. Cells were labeled with 3~Pi
`(A) or [3jS]cysteine (Bl in the presence or absence of MAb 40 5 (150
`nM) for 11 h. Samples were prepared and separated as described in
`Materials and Methods. The autoradiogram shown here was ob(cid:173)
`tained by 6 h of exp0sure. (Cl Cells were labeled with [35S]cv ~teine
`for 11 h in the presence of MAb 405 (150 nM. lane 2) or F(ab) <400
`nM . lane 3) or with culture medium (lane 1). Samples were prepared
`and immunoprecipitation was carried out as described in Materials
`and Methods. An autoradiogram of a dried gel is shown here.
`Quantitation of the p185H£Rl bands was obtained by densitometric
`scanning (A through C) or by determining radioactivity associated
`with bands (A and 8 ). Quantitation by determining the radioactivity
`associated with pl85H£R1 bands in panels A and B gave results
`similar to tho~e with densitometric scanning, and there was a 27% :!:
`3% additional red11c1infl in 32P-labeled pl85H£Rl compared with
`"S-labeled pl85H£Hl . ID) lmmunoblotting of pl85H£Rl and EGF-R
`proteins. In experiment 1 (Exp. I). 32P-labeled SK-BR-3 cell extracts
`(50 µ.g of pMteinl used in Fig. 2. lanes 1 and 2. were resolved on a
`7"K SDS-p0lyacrylamide gel and then immunoblotted with anti-Pl85
`M:\b 9G6 or aati-EGF-R p0lyclonal antibody RK-11. Experiment 2
`~hows the immunoblotting of unlabeled SK-BR-3 cell extracts
`µrepared following culture for 15 h with or without 30 nM MAb 405.
`Since some of the extracts used here were radiolabeled. immuno(cid:173)
`blotted membranes were visualized by using a protein A-gold
`..:11ru1ni:ement kit i30). Abs. AntibvJu:s.
`
`down-regulation of receptor protein. First, we determined
`what fraction of the 35S-labeled pl8511ERz is present on the
`cell surface at J7°C (Fig. 4A). In these experiments.
`pl8511£Rz expressed on the plasma membrane was identified
`by its capacity to bind MAb 405 prior to cell lysis. The
`results indicate that 19% ± 4% (average from three different
`experiments) of total 35S-labeled pl 8511ER2 is expressed on
`the cell surface under these experimental conditions; thus
`pl8511£Rz is available for down-regulation by MAb 405.
`Down-regulation of EGF-R has been shown to be dependent
`on temperature (41). To confirm that down-regulation of
`surface pi85HERZ also is reduced at 4°C, experiments were
`performed to analyze the effect of temperature on the
`abundance of 35S-labeled pl85HERi on the cell surface.
`Results indicated that at 4°C the amount of total 35S-labeled
`pl8511£Ri expressed on the surface increased to J5% ± J%
`(data not shown) compared with 19% ± 4% of total 35S(cid:173)
`labeled pl8511ER~ at J7°C.
`In order to defi ne the contribution of down-regulation to
`MAb-induced reduction in pl85 11£Rz phosphorylation, we
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 3 of 8
`
`3 of 8
`
`Celltrion, Inc., Exhibit 1088
`
`

`

`Antibody P185
`
`MOL. CELL. BIOL.
`
`P-Tyr
`
`11 21 311
`
`1 2 3
`
`.. -
`
`-
`
`FIG. 5. Partial agonist nature of F(ab). Subconftuent SK-BR-3
`cells were labeled with 32P; for 15 h. Some cultures were treated with
`400 nM F(ab) for the indicated times. The cells were lysed in 800 µ.I
`of extraction buffer. The lysates were divided into two equal parts of
`350 µ.I each and then immunoprecipitated with MAb 906 (lanes 1 to
`3) or with antiphosphotyrosine MAb PY-69 (lanes 1' to 3'). An
`autoradiogram resulting from a 1-h exposure of dried gel is shown
`here. Lane 1, Control; lane 2. F(ab) incubation for 15 min; lane 3,
`F(ab) incubation for 60 min.
`
`982
`
`KUMAR ET AL.
`
`A. EXP. 1
`I 1 2 1
`
`EXP. 2
`I 3 4 1
`
`B.
`
`4°C
`37°C
`2 3 11 4 5
`
`405
`
`- +
`- +
`FIG. 4. (A) Quantitation of surface expression of ' 5S-labeled
`pl85HERZ. Cells were labeled with C' 5S]cysteine for 11 h. At the end
`of incubation, some cultures were lysed in 500 µ.I of lysis buffer for
`the determination of total 35S-labeled p185HERZ by immunoprecipi(cid:173)
`tation with 10 µ.g of 405 (Materials and Methods). For measuring the
`surface expression of 35S-labeled p185HERZ. cultures were washed
`with phosphate-buffered saline and further incubated with F-12/
`OMEM-20 mM HEPES (pH 7 .5) containing 20 µ.g of high-affinity
`MAb 405 per ml for 1 hat 4°C. The cultures were washed. lysed in
`500 µ.I of extraction buffer, and processed for immunoprecipitation
`by adding RAM-protein A-Sepharose beads but no more MAb 405
`during the immunoprecipitation procedure. The results of two
`representative experiments are shown here. Lanes 1 and 3, Total
`35S-labeled pl85H£RZ; lanes 2 and 4, 35S-labeled pl85H£Ri on the cell
`surface. (B) Effect of MAb 405 on surface expression of pl85HERZ in
`a temperature shift experiment. SK-BR-3 cells were first equili(cid:173)
`brated with 32P; for 4 h at 37°C (lane 1) and then further incubated
`with or without MAb 405 for an additional 11 h (in the continuous
`presence of 32P;) either at 37°C (lanes 2 and 3) or at 4°C {lanes 4 and
`5). The autoradiogram shown here was obtained by exposing lanes
`1 to 3 for 24 h and lanes 4 and 5 for% h. Quantitation of the amount
`of n p associated with pl85HERZ bands was obtained by densitomet(cid:173)
`ric scanning of the autoradiogram and by determining the radioac(cid:173)
`tivity associated with p185HERZ bands (A and B).
`
`analyzed the effect of incubation with MAb 405 at 4°C. In
`these studies, cells were first equilibrated with 32P1 for 4 h at
`37°C (Fig. 4B, lane 1) and then maintained at 37°C (lanes 2
`and 3) or shifted to 4°C (lanes 4 and 5) for an additional 11 h ,
`with or without MAb 405. A comparison of the labeled
`material in lanes 2 and 4 in Fig. 4B (ftuorographs exposed for
`24 and % h, respectively) showed a significant 85% reduc(cid:173)
`tion in 32P labeling of pl85HERi during 11 h at 4°C compared
`with labeling at 37°C. However, incubation of cells at 4°C did
`not prevent a further substantial MAb-mediated reduction in
`steady-state levels of pl85HERi phosphorylation: there was a
`34% decrease at 4°C (compare lanes 4 and 5) and a 51%
`decrease at 37°C (compare lanes 2 and 3). Taken together,
`these observations indicate that MAb-induced reduction in
`pl85HER phosphorylation cannot be completely accounted
`for by down-regulation.
`Experiments were performed to determine whether the
`F(ab) fragment might have the capacity to act as an agonist
`by activating tyrosine phosphorylation. The results in Fig. 5
`indicate that the addition of F(ab) for 15 min sli_ghtly stimu(cid:173)
`lated in vivo tyrosine phosphorylation of pl85 E R i in cul(cid:173)
`tures labeled with 32P (Fig. 5, lane 2'). However, there was
`no activation in cultures exposed to F(ab) for a longer
`treatment of 60 min (Fig. 5, lane 3 '). The observation that the
`F(ab) fragment of 405 does not down-regulate the 35S-
`
`labeled pl85HERi but can act for a short time as a partial
`agonist is interesting; however, we have not attempted to
`further characterize these properties in the present study.
`Activation of phosphorylation of p18SHER2 in presence or
`absence of newborn calf serum. Next, we investigated the
`that might stimulate
`possible source of the factor(s)
`pl85HERi phosphorylation. As shown in Fig. 6A, culturing
`the cells in serum-free medium resulted in a steady-state
`level of phosphorylation of p18511ER2 reduced 56% (lane 1)
`compared with that observed in the continuous presence of
`newborn calf serum (lane 3). The addition of MAb 405 in
`serum-free culture conditions further reduced pl85HERi
`
`B.
`
`+ +
`
`A.
`
`2 3 4
`
`-.....
`C. •
`
`Serum
`405
`
`+ +
`+
`
`+
`
`3
`
`2
`FIG. 6. (A) Detection of newborn calf serum-mediated phos(cid:173)
`phorylation of pl85HERi. Subconfluent SK-BR-3 cells were labeled
`with 32P; in the culture medium without (lanes 1 and 2) or with (lanes
`3 and 4) 5% newborn calf serum for 15 h. Cultures analyzed in lanes
`2 and 4 also were continuously exposed to 150 nM MAb 405.
`Samples were prepared and immunoprecipitated for assaying the
`amount of pl85HERi as described in the Materials and Methods.
`Quantitation of the pl85 bands was obtained by densitometric
`scanning of the autoradiogram. (B) Control experiment showing
`effect of serum on the 32P; labeling of EGF-Rs for 15 h in SK-BR-3
`cell cultures. Cell extracts were immunoprecipitated with anti(cid:173)
`EGF-R MAb 528, which recognizes one distinct band with an
`approximate molecular mass of 170 kDa (arrow). (C) Two-dimen(cid:173)
`sional thin-layer electrophoresis pattern of 32P-phosphoamino acids
`in a hydrolysate of the p185HERZ immunoprecipitated in panel A. S,
`Phosphoserine; T . phosphothreonine; Y. phosphotyrosine. Number
`at lower left of each autoradiogram indicates the following culture
`conditions: l , with no serum; 2, with serum; 3, with serum and MAb
`405. Tyrosine phosphorylation in control cells was visualized
`faintly on the autoradiogram but reproduces poorly.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1088 Page 4 of 8
`
`4 of 8
`
`Celltrion, Inc., Exhibit 1088
`
`

`

`VOL. 11, 1991
`
`MODULATION OF PHOSPHORYLATION OF pl85HER1
`
`983
`
`phosphorylation (Fig. 6A, lane 2) to 20% of the steady-state
`levels achieved in the absence of newborn calf serum (Fig.
`6A, Jane 1). Experiments were done to examine the capacity
`of newborn calf serum to stimulate tyrosine phosphorylation
`ofp185H£Rl in short-term ~eatment. There was no increased
`activation of phosphorylation when serum-free cultures
`were supplemented with newborn calf serum for 30 min at 37
`or 4°C (data not shown).
`To determine the specificity of the ca~city of newborn
`calf serum to stimulate activation of p185 .ERZ phosphoryla(cid:173)
`tion in SK-8R-3 cells, we investigated the potential for
`serum activation of another closely related molecule, the
`EGF-R. There was no potentiating effect of newborn calf
`serum on phosphorylation of the EGF-R in SK-8R-3 cells
`(Fig. 68).
`Ravi~ shown an increase in the steady-state levels of
`p185H£
`phosphorylation induced by newborn calf serum
`and its reduction by MAb 405, we determined the phos(cid:173)
`phoamino acid content of p185H£Ri under these conditions
`by two-dimensional thin-layer electrophoresis (Fig. 6C).
`p185HER2 from Cells Cultured in the absence Of newborn Calf
`serum contained predominantly pbosphoserine and phos(cid:173)
`photlireonine with little phosphotyrosine (Fig. 6C, blot 1).
`The presence of some phosphorylatio~ on tyrosine can be
`demonstrated by longer exposure of the autoradiogram but is
`not visualized well in the figure shown. Quantitation of the
`relative amount of label in each amino acid was obtained by
`scraping the ninhydrin-ideiltified spots from the thin-layer
`plate for liquid scintillation counting. Activation by 5%
`newborn calf serum increased the total phosphoamino acid
`content 2.2-fold, while for phosphotyrosine, the increase
`was 3.9-fold (Fig. 6C, blot 2). Inhibition of newborn calf
`serum-mediated stimulation ofpl85H£Ri phosphorylation by
`MAb 405 resulted in a parallel reduction in the content of all
`three phosphoamioo acids (Fig. 6C, blot 3).
`Tyrosine phosphorylation or pl858
`ER2 . To further quanti(cid:173)
`tate tlie relative increase in the steady-state phosphotyrosine
`content of pl85H£RZ induced by newborn calf serum, cells
`were metabolically labeled with [35S]cysteine and assayed
`for the steady:state phosphotyrosine content of p185HERl by
`using antiphosphotyrosine MAb PY-69. MAb PY-69 was
`specific for pbospbotyrosine in the immunoprecipitation
`reaction; i.e. , we were able to show competion for binding
`with cold phosphotyrosine and not with phosphoserine in
`experiments with 35S-labeled EGF-R (data not shown). As
`illustrated in Fig. 7 A, lanes 1' to 4', the amount of phosphor(cid:173)
`ylation of p185HERi on tyrosine increased with the concen(cid:173)
`tration of newborn calf serum in the culture medium. The
`level of activation of tyrosine phosphorylation in serum-free
`medium was 18% of that observed in 10% newborn calf
`serum , and in medium containing 2.5% serum, tyrosine
`phosphorylation was 35% of that observed with a serum
`concentration of 10%. As a control, equal amounts of labeled
`cell extracts were immunoprecipitated with anti-pl85HER2
`MAb 9G6 (Fig. 7 A, lanes I through 4). There was no
`significant effect of serum on the levels of 35S-labeled
`pl85H£RZ in the cells. To quantitate these results, the ratios
`of phosphotyrosine-associated counts to total counts asso(cid:173)
`ciated with p1858 £R2 are presented in Fig. 78, which shows
`a dose-dependent increase of up to 5.4-fold with 10% new(cid:173)
`born calf serum in the culture medium.
`Partial depletion of activating factor from newborn calf
`serum. Since our results indicated the presence of some
`activating factor(s) for p185H£R2 phosphorylation in new(cid:173)
`born calf serum, we sought confirmation of this observation
`by determining whether newborn calf serum could be de-
`
`A.
`
`Antibody
`
`-
`
`P-Tyr
`2'
`3'
`
`4'
`
`,.
`
`Pl85
`2
`3
`
`4
`
`-
`
`% Serum
`
`O
`
`2.5
`
`5
`
`10
`
`0
`
`2 .5
`
`5
`
`10
`
`B.
`
`500
`
`1()-
`
`ro E a:c
`_u
`...... 0 300
`~a
`d.~
`100
`
`•
`
`/
`
`/
`
`C . 1 2 3 4
`
`-
`
`0
`
`10
`
`5
`Percent Serum, VIV
`FIG. 7. (A) Detection of tyrosine-specific phosphorylat.ion of
`pl85HERz by newborn calf serum. Subconfluent SK-BR-3 cells were
`metabolically labeled with [35S]cysteine iii the presence of dilferent
`concentrations of serum for 15 h. Cells were lysed in 650 µI of
`extraction bu1fer. The lysates were divided into two equal parts of
`300 µ.I each and then immunoprecipitated with anti-pl85HER1 MAb
`9G6 (lanes 1 to 4) or antiphosphotyrosine MAb PY-69 (lanes l ' to
`4'). Other details of the assay were as described in the legend to Fig.
`5. To detect the phosphotyrosine (P-Tyr) signal in cells cultured in
`the absence of serum (lane l '), it was necessary to expose the
`autoradiogram for 20 h, which resulted in overexposure of lanes 1 to
`4. (B) To quantitate the data in panel A, the radioactivity associated
`with p185HER1 was determined by counting the excised bands in a
`liquid scintillation counter. The ratios of counts in pl85H£Rz phos(cid:173)
`photyrosine over total counts in pl85H~ were plotted as a percent(cid:173)
`age of control with 0% newborn calf serum against the concentration
`of newborn calf serum used in the culture medium. (C) Depletion of
`the activator(s) of p18SHER1 phosphorylation in serum. SK-BR-3
`cells were labeled with 32P1 in the absence or presence of serum for
`15 h. Lane l, Control without serum; lane 2, medium with 5%
`newborn calf serum; lane 3, medium with 5% newborn calf serum
`depleted of factor(s) by three repetitive adsorptions of 4 h each on
`SK-BR-3 cells at 4"C; lane 4, control medium adsorbed on A431
`cells. Cell extracts were prepared and p185HER1 was assayed as
`described in Materials and Methods.
`
`pleted of such a factor(s). In these experiments, phosphate(cid:173)
`free medium containing 5% newborn calf serum was treated
`by repetitive absorption with SK-8R-3 cells (three times, for
`4 h each time, at 4°C). Culture medium treated in an identical
`manner by adsorption with A431 cells, which do not express
`high levels of pl85"£R2, was used as control. Figure 7C
`shows that there was a 53% reduction in p1858 £R2 phosphor(cid:173)
`ylation in SK-8R-3 cells cultured in the presence of medium
`preadsorbed with SK-8R-3 cells (lane 3) compared with
`untreated medium (lane 2), and there was only a 16%
`reduction in p185HERi phosphorylation when SK-8R-3 cell
`cultures were supplemented with medium preadsorbed with
`A431 cells (Fig. 7C, lane 4).
`Activation factor(s) In newborn calf serum was not TGF-o
`or EGF. pl85H£Ri has been shown to be phosphorylated
`when EGF-Rs are activated by ex~sure to EGF or TGF-a,
`which are not ligands for pl8588
`(24, 40). To determine
`whether the newborn calf serum-mediated 2.2-fold enhance(cid:173)
`ment of pl85HER2 phosphorylation resulted from the acti